Studies on asexual stage immunity to P. falciparum 1. Evaluate the merozoite antigen PfRH5 as a vaccine candidate. Previously we have collaborated with investigators in LIG to show that antibodies to RH5 isolated from those living in Mali are able to inhibit parasite invasion of red cells (GIA activity), lending support to this protein as a new vaccine candidate. Interestingly, Malian sera has very low levels of antibodies to this antigen. Our collaborators at Oxford University are pursuing this protein as a vaccine candidate and,as a prelude to that, have conducted an immunization-challenge trial in Aotus monkeys. We have collaborated on evaluating GIA activity in sera from the monkeys with PfRH5 and these results have been published; the promising results have supported proceeding to a human clinical trial which has recently been completed and we are currently analyzing these sera. Also we have evaluated the GIA activity of antibodies to both RH5 and the RIPr protein (RH5 interacting protein) which appear together on the surface of the invading merozoite. Each protein elicits strong GIA activity 2. Investigate other targets of merozoite immunity - AMA1 While AMA1 is a prominent vaccine candidate, it has two issues:1) antigenic polymorphism so that antibody responses tend to be strain-specific and 2) insufficient antibody production to result in protective immunity in humans. Our data as well as that of our collaborators supports the use of a mixture of 4-5 different AMA1 alleles to overcome the polymorphism and elicit broadly reactive antibodies. Further, we have collaborated with LMVR investigators (Dr. Louis Miller) to show that using AMA1 in conjunction with its partner protein RON2 produces antibodies with greater activity in a standardized parasite growth inhibition assay (GIA). This complex is required for triggering junction formation between the merozoite and the red cell surface. Evaluation of this combination in an immunization-challenge trial in a non-human primate (Aotus) has been conducted and we have provided supportive GIA data for a human clinical trial. 3. Studies of immunity to malaria in Kenieroba, Mali. Our 4-year investigation of the acquisition of immunity to malaria in Malian children represents perhaps the most detailed longitudinal study of malaria in African children that has been conducted, and it details the critical impact of the sickle cell trait (HbAS) in protection against malaria. We have previously evaluated the antibody responses of these children in several different assays including ELISA,GIA, and antibodies to variant surface antigens on malaria-infected red cells (VSA). In 2014-2015 we collaborated with others to broaden the scope of anti-parasite functional activities and have added the neutrophil-dependent antibody-dependent respiratory burst assay. A manuscript on this is in press. 4. We continue to work with Dr. Amy Bei and Dr. Dyann Wirth of Harvard University, who are studying changes in frequency of different P.falciparum clones over time in Senegal. We have worked with them to examine the influence of human immune responses on the changes in clonal parasite patterns, particularly related to the observation of parasites with common genetic signatures. The first results of these studies using GIA and the VSA assay were published this year and an expanded study is in progress. Studies on parasite sexual stages and malaria transmission: 1.) Develop quantitative methodology for analysis of the standard mosquito membrane feeding assay (SMFA) to evaluate transmission blocking activity and extend this to parasites in the field. The gold standard assay to evaluate the ability of antibodies to block transmission to mosquitoes is the SMFA, and we have performed an in depth study of this assay to define its characteristics in order to have confidence in assessing potential transmission blocking vaccine (TBV) candidates. We have shown that the SMFA is quite reproducible at high concentrations of antibody but highly variable at low concentrations and we have worked with the Biostatistics group at NIAID to develop a computer model of the assay. In 2014-2015 we have extended our collaboration with this group to show that we can predict the impact of a specific antibody on the reduction of P. falciparum parasite prevalence in mosquitoes from the reduction in oocysts in an SMFA and from the number of oocysts in the control mosquitoes. This is important because reduction in malaria prevalence in mosquitoes by an antibody is key to reducing transmission. 2.) Search for and evaluate new possible transmission blocking vaccine candidates. Using SMFA in 2014-2015 we have evaluated a number of potential transmission blocking vaccine candidates. In collaboration with colleagues at Oxford University we have produced and tested a number of known and unknown sexual stage proteins from P. falciparum. These proteins have been produced in HEK293 cells and the products have been used to immunize mice. Sera from these animals have been evaluated in the SMFA but none have shown high levels of oocyst inhibition in this assay. 3.) Assess the presence of asexual and sexual stage parasites in residents of Kenieroba, Mali throughout the year. In the spring of 2013 we initiated a new study (NIH 13-I-N107) of malaria transmission to address the limited information on transmission in malaria endemic areas, and in 2014 we completed the field aspect of this study. Volunteers representing all age groups were finger pricked twice per month for 1 year to collect DNA and RNA. During the 2014-2015 year we completed analysis of over 10,000 samples of parasite DNA on filter paper. In October 2013 (middle of the wet season), P. falciparum prevalence in the cohort was 48.0%, and in March 2014 (middle of the dry season), the prevalence dropped to 18.0%. We also analyzed the longitudinal prevalence of the cohort; we observed that the 9-12 years old age group had the highest median longitudinal prevalence (38.1%) compared to the other age groups, and males had a higher median (33.3%) than females (22.0%). By a linear regression model, only age and gender showed significant effects on the longitudinal prevalence (p<0.0001 and p=0.0008, respectively), while other host factors did not. There are few studies of longitudinal prevalence of parasites and we are preparing these results for publication. 4.) Using the RNA collections from the same study, we are using a qRT-PCR procedure to identify Pfs25 mRNA specific for gametocytes. In 2014-2015 we have completed part of the RNA analysis; these results will allow us to identify those individuals capable of malaria transmission who might be targeted for intervention. 5.) Determine whether Kenieroba residents develop antibodies which have blocking activity in SMFA. This study of transmission includes a venipuncture sample from all the volunteers 3 times during the year. These larger volume serum samples are being used in SMFA to determine whether antibodies have transmission blocking activity, particularly after the rainy season. 6.) With support from PATH/MVI, we have begun to evaluate humanized monoclonal antibodies to 3 different sexual/mosquito stage antigens. 7.) We have initiated studies to examine the differentiation pattern of P. falciparum gametocytes in culture for 3 weeks using RNASeq techniques. This will be extended to transcripts produced in infected mosquitoes.